A novel type of AIE material as a highly selective fluorescent sensor for the detection of cysteine and glutathione

Abstract

A facile and efficient method was developed for the synthesis of isothiazolo[5,4-b]pyridin-3(2H)-one derivatives. This new type of AIE materials was studied through photophysical analysis, X-ray and theoretical calculations. Then, based on a ring-opening reaction, we found a useful application of this material in cysteine and glutathione detection.

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Introduction

AIE (aggregation-induced emission) materials were first reported by Tang's group at 2001.¹ This new kind of luminophore has plenty of advantages; unlike traditional photophysical materials, the fluorescence emission is enhanced rather than quenched after aggregation.

Traditional fluorescent compounds have large π-conjugated frameworks like plates. When molecules aggregated, the fluorescence quenched. It is widely known as aggregation-caused quenching (ACQ) effect, this effect greatly limits the practical application of luminescent materials. To avoid ACQ effect, two common methods were used: adding big blocking groups or diluting the molecules in electricity conducting polymers. But the methods either increased the synthetic difficulty or reduced the efficiency. Different from the traditional fluorescent materials, AIE luminophores were proved to be the promising materials to eliminate ACQ (aggregation-caused quenching) effect. These materials have been wildly used in all kinds of areas, such as organic lighting-emitting diodes (OLEDs),² chemosensors (CO₂, HSO₃⁻ detection),³ bioprobe (DNA, RNA analysis)⁴ and biomedical applications.⁵

We have been focusing on the development of facile synthesis of AIE molecules using tandem reactions.⁶ Novel kind of AIE materials was synthesized and applied as cysteine and glutathione bioprobes. Cysteine and glutathione play important roles in many diseases such as liver damage, cancer and HIV infection.⁷ Cysteine is one of the most important amino-acids to human body, it is a precursor not only for a lot of proteins but also for glutathione.⁸ And glutathione levels are key index to evaluate the detoxification and redox state in organisms. High sensitive and selective fluorescent cysteine and glutathione probes are extraordinary needed in bio-research.⁹ As a result, several fluorescence probes were designed based on different detection mechanisms. Zhang et al.¹⁰ developed a near-infrared probe based on the conjugate addition–cyclization reaction and it can be applied in living cells. Zhou et al.¹¹ reported a cysteine probe promoted by respond-assisted electrostatic attraction. Lee et al.¹² designed an aryl-thioether substituted nitrobenzothiadiazole probe for the detection of cysteine and homocysteine.

Herein, we utilized thiosalicylic acids reacted with different amines to form new kinds of AIE materials. The reaction was processed under mild conditions, and high yields of products were obtained.

Results and discussion

As shown in Fig. 1, four different compounds were obtained through one-pot tandem reaction. Solid-state fluorescent imagines were shown in Fig. 1.

Fig. 1 Fluorescence microscope images (ISO 200) of compound 3a–c.

Different crystal forms were observed. All the molecules showed good optical properties except 3d which was proved to be a colorless oil. So we speculated that forming proper crystal structure was essential to lumine. Meanwhile, compound 3b showed the best fluorescence quantum yield (18%). The fluorescence quantum yields of 3a–d in THF were also detected: 3a (99%), 3b (9.0%), 3c (2.8%) and 3d (99%) (Fig. 2).

Fig. 2 X-ray structure of compound 3a.

X-ray crystallographic analysis was shown in Fig. 3. It configured the structure of 3a. Meanwhile, HOMOs/LUMOs of 3a–d were also calculated by Gaussian 03 program using the B3LYP method with the 6-31G basis set (Scheme 1).¹³ Clear intramolecular charge transfer (ICT) effects were observed in 3b and 3d. As a result, 3b gave better luminescence than the other molecules. Compound 3d was oil under room temperature, which made 3d lose AIE characteristic (Fig. 4).

Fig. 3 Fluorescence emission spectra of 3a–d (10 μM) in THF/water (1/99, v/v).

Scheme 1 Synthesis and absolute quantum yields determined by a calibrated integrating sphere system of fused isothiazolidin-3-ones 3.

Fig. 4 HOMO/LUMO imagines of 3a–d calculated by Gaussian 03 program using the B3LYP method with the 6-31G basis set.¹³

Compound 3b showed the best optical characteristics. The DLS (Dynamic Light Scattering) radiuses of 3a–c were also measured (Fig. S3 in the ESI†). The results confirmed that compound 3a–c were aggregated into sub-micron particles in THF/water solutions.

What is more, the AIE properties of 3a and 3b were determined in THF/water (from 100/0 to 1/99, v/v) shown in Fig. 5 and 6. An interesting optical phenomenon was found. The compounds showed ACQ effect at first, then AIE effect was observed after adding large amount of water. In pure THF, molecule 3b gave luminescence at 400 nm. Then the fluorescence quenched after water fraction raised from 0% to 60%. When water fraction increased to 90%, the molecules began to aggregate into sub-micron particles. After that, a new fluorescence emission was observed at 450 nm. The emission was both bathochromic-shifted and enhanced after aggregation.

Fig. 5 Fluorescence emission spectra of 3a (excitation at 330 nm) in THF/water mixtures with different water fractions (f_w).

Fig. 6 Fluorescence emission spectra of 3b (excitation at 330 nm) in THF/water mixtures with different water fractions (f_w).

After all the photophysical measurements were taken, we developed compound 3b into the cysteine and glutathione bioprobes based on a ring-opening reaction. The detection system was a THF/water solution buffered by 20 mM HEPES at pH 7.2. The probes were existed as sub-micron particles in the system. They were highly fluorescent at first. After the addition of cysteine, the fluorescence intensity decreased dramatically. It was a highly selective analysis system.

The selectivity of probe 3b for Cys (cysteine) and GSH (glutathione) was compared to the other amino acids, such as Gly (glycine), Ala (alanine), Leu (leucine), Val (valine), Pro (proline), Met (methionine), Phe (phenylalanine), Trp (tryptophan), Ser (serine), GSSG (oxidized glutathione) shown in Fig. 7. Only cysteine and GSH (glutathione) could make the probe 3b quench. The analytical system was proved to be highly selective.

Fig. 7 Fluorescence emission spectra (excitation at 330 nm) of 100 μM 3b in THF/water (v/v, 1/99) mixtures buffered by 20 mM HEPES at pH 7.20 with 20 equiv. Cys, Gly, Ala, Leu, Val, Pro, Met, Phe, Trp, Ser.

Fluorescence titration of cysteine detection was taken in THF/water (1/99, v/v) buffered by 20 mM HEPES. The probe solution (3b, 1 × 10⁻⁵ M) was prepared according to the methods described in the ESI.† Then the titration was proceeded by adding cysteine solution (1 × 10⁻³ M, 10 μL each time) into the probe solution (1 × 10⁻⁵ M, 1 mL). Upon the addition of cysteine, the emission peak gradually decreased.

The fluorescence emission curve was shown in Fig. 8 at pH 7.20. And titration linear for Cys at the concentration between 0 μM and 7 μM was shown in Fig. 9 (R = 0.99987, n = 7). The linear regression equation was calculated to be 1 − I/I₀ = −0.000129916 + 0.12986X. According to IUPAC, the detection limit was calculated as 0.5 μM and a relative standard deviation of 0.9% was measured for 5.0 μM Cys. And the fluorescence titration of GSH (glutathione) was also proceeded under the same condition. The result was shown in Fig. 10.

Fig. 8 Fluorescence emission (excitation at 330 nm, slit: 5 nm/10 nm) of 3b (1 × 10⁻⁵ M) was titrated with Cys (0–0.9 equiv.) in THF/water solution (1/99, v/v) buffered by 20 mM HEPES at pH 7.2.

Fig. 9 Calibration graph for cysteine. Working conditions: pH 7.2, reaction time 90 s.

Fig. 10 Fluorescence emission (excitation at 330 nm, slit: 5 nm/10 nm) of 3b (1 × 10⁻⁵ M) was titrated with GSH (0–0.9 equiv.) in THF/water solution (1/99, v/v) buffered by 20 mM HEPES at pH 7.2.

Conclusions

A series of AIE compounds were synthesized through one-pot tandem reaction. Different optical properties were observed when R group changed. Only the materials which have solid-state showed good AIE characteristics. An interseting bathochromic-shifted and enhanced luminescence were also detected. At last, we developed a new kind of cysteine and glutathione probes based on a ring-opening reaction. The detection system was proved to be highly selective.

Experimental section

All reagents and solvents were pure analytical-grade materials purchased from commercial sources and were used without further purification, if not stated. All reactions were monitored by thin-layer chromatography (TLC). ¹H NMR spectra were recorded on a Bruker Avance 400 or 300 spectrometer at 400 or 300 MHz, using CDCl₃ as solvent and tetramethylsilane (TMS) as internal standard. ¹³C NMR spectra were run in the same instrument at 100 or 75 MHz. HRMS spectra were determined on a Q-TOF6510 spectrograph (Agilent).

To a solution of 2-mercaptonicotinic acid (0.31 g, 2 mmol) in CH₂Cl₂ (20 mL) thionyl chloride (0.3 mL) was added, the solution was refluxed for 2.5 h (oil bath). The mixture was evaporated in vacuo, and the solvent and thionyl chloride were removed, yellow solid was obtained. The yellow solid was dissolved by CH₂Cl₂ (20 mL), then cyclohexylamine (0.20 g, 2 mmol) dissolved in 10 mL CH₂Cl₂ was added dropwise to the ice bath solution. The mixture was stirred overnight, then H₂O (100 mL) was added and the mixture was filtered and extracted with CH₂Cl₂ (3 × 50 mL). The combined organic layers were washed by chilled water (3 × 100 mL), dried by Na₂SO₄, filtered, and evaporated in vacuo. The crude product was purified by flash chromatography on silica gel (hexane/EtOAc = 5 : 1). Compound 3a was obtained as white plate crystals (0.11 g, 47%).

2-Cyclohexylisothiazolo[5,4-b]pyridin-3(2H)-one (3a)

White solid (47%). ¹H NMR (300 MHz, CDCl₃) δ 8.73 (dd, 1H, J = 4.5, 1.5 Hz), 8.29–8.26 (m, 1H), 7.37–7.32 (m, 1H), 4.69–4.59 (m, 1H), 2.07 (d, 2H, J = 11.4 Hz), 1.90 (d, 2H, J = 13.2 Hz), 1.74 (d, 1H, J = 13.2 Hz), 1.65–1.40 (m, 4H), 1.30–1.16 (m, 1H). ¹³C NMR (75 MHz, CDCl₃) δ 163.1, 162.4, 153.2, 134.7, 120.6, 120.3, 53.2, 32.9, 25.6, 25.2. HRMS calcd for C₁₂H₁₄N₂OS, 235.0900; found, 235.1052.

2-(3,4,5-Trimethoxyphenyl)isothiazolo[5,4-b]pyridin-3(2H)-one (3b)

Yellow solid (43%). ¹H NMR (300 MHz, CDCl₃) δ 8.82 (dd, 1H, J = 4.8, 1.5 Hz), 8.34 (dd, 1H, J = 7.8, 1.5 Hz), 7.42 (dd, 1H, J = 8.0, 1.5 Hz), 6.91 (s, 1H), 3.90 (s, 6H), 3.88 (s, 3H). ¹³C NMR (100 MHz, CDCl₃) δ 162.6, 161.9, 154.1, 153.6, 137.6, 135.2, 132.0, 121.1, 119.7, 103.1, 61.0, 56.3. HRMS calcd for C₁₅H₁₄N₂O₄S, 319.0708; found, 319.0751.

Methyl 2-(3-oxoisothiazolo[5,4-b]pyridin-2(3H)-yl)benzoate (3c)

White solid (45%). ¹H NMR (300 MHz, DMSO-d₆) δ 8.94 (dd, 1H, J = 7.6, 1.6 Hz), 8.38 (dd, 1H, J = 8.0, 1.6 Hz), 7.98 (dd, 1H, J = 8.0, 1.6 Hz), 7.83–7.78 (m, 1H), 7.67–7.59 (m, 3H), 3.64 (s, 3H). ¹³C NMR (100 MHz, CDCl₃) δ 165.0, 162.7, 161.9, 154.6, 135.3, 134.5, 133.6, 131.0, 129.7, 129.4, 129.2, 121.7, 117.9, 52.3. HRMS calcd for C₁₄H₁₀N₂O₃S, 287.0446; found, 287.0551.

2-(Naphthalen-2-yl)isothiazolo[5,4-b]pyridin-3(2H)-one (3d)

Colorless oil (38%). ¹H NMR (300 MHz, DMSO-d₆) δ 8.98 (dd, 1H, J = 4.6, 1.6 Hz), 8.45 (dd, 1H, J = 7.8, 1.5 Hz), 8.16–8.10 (m, 2H), 7.78 (d, 1H, J = 6.6 Hz), 7.70–7.56 (m, 5H), 3.31 (s, 1H). ¹³C NMR (100 MHz, CDCl₃) δ 163.4, 162.7, 155.1, 135.9, 134.4, 132.4, 130.6, 129.0, 128.5, 128.0, 127.4, 126.2, 123.1, 122.3, 118.6. HRMS calcd for C₁₆H₁₀N₂OS, 279.0547; found, 279.0579.

Acknowledgements

We are grateful to the National Science Foundation of China (Grant nos 21172131, 21273131) and State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College (no GTZK201405) for financial support of this research.

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Footnote

† Electronic supplementary information (ESI) available. CCDC 1051210. For ESI and crystallographic data in CIF or other electronic format see DOI: 10.1039/c5ra06244f

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